This invention is in the field of molecular biology and medicine. More specifically, it relates to novel vector constructs and to methods of making DNA constructs for introducing targeted mutations into embryonic stem cells.
A major challenge facing biologists today is determining the function of over half a million partial cDNA sequences of various genes, known as expressed sequence tags (ESTs), that are publicly available. In most cases the function of the full-length genes represented by the ESTs remains unknown. Thus, the ability to determine function of these gene sequences is important for disease diagnosis, prediction, prevention and treatment
In recent years, mouse geneticists have succeeded in creating transgenic animals by manipulating the genes of developing embryos and introducing foreign genes into these embryos. Once these genes have integrated into the genome of the recipient embryo, the resulting embryos or adult animals can be analyzed to determine the function of the gene.
U.S. Pat. Nos. 5,464,764 and 5,487,992 describe one type of transgenic animal in which the gene of interest is deleted or mutated sufficiently to disrupt its function. These xe2x80x9cknock-outxe2x80x9d animals are made by taking advantage of the phenomena of homologous recombination. (See, also U.S. Pat. Nos. 5,631,153 and 5,627,059). Briefly, conventional targeting DNA vectors contain (1) two blocks of DNA sequences that are homologous to separate regions of the target site; (2) a DNA sequence that codes for resistance to the compound G418 (Neor) between the two blocks of homologous DNA (i.e. positive selection marker) and (3) DNA sequences coding for herpes simplex virus thymidine kinases (HSV-tk1 and HSV-tk2) outside of the homologous blocks (i.e. negative selection marker). When this vector is introduced into the embryonic stem cell, homologous recombination inserts the Neor gene into the target genome, disrupting function of that gene.
The production of constructs useful in producing knock-out animals is a time and labor intensive process. (See, e.g., U.S. Pat. No. 5,464,764) First, genomic clones must be isolated by screening a genomic library with a radioactive probe. To isolate an individual clone requires multiple screens and can take more than 3 weeks. Once the clone is isolated, a restriction map is created in order to aid in the identification of fragments flanking the gene of interest. Again, this process can take several weeks. Finally, the flanking sequences are cloned into the targeting vector. Even in methods which make use of polymerase chain reaction techniques, a partial restriction map of the gene locus is created. (See, Randolph et al. (1996) Transgenic Research 5:413-420). In all, using conventional techniques, production of a DNA targeting construct can take several months.
The present invention provides novel constructs (e.g., plasmid vectors) useful in a rapid and efficient method for generating DNA constructs suitable for introduction into embryonic stem cells. The novel methods described herein eliminate the need for the traditional hybridization isolation of a single genomic clone, restriction mapping of the clone and multiple cloning steps. Thus, the present invention provides an unexpected reduction in the time required for making a xe2x80x9cknock-outxe2x80x9d vector. Methods described in the art require 2 to 4 months to accomplish what the claimed invention can achieve within 1-2 weeks.
The unexpected increase in efficiency accomplished by the methods described herein involves methods that have not previously been applied to the process of making a xe2x80x9cknock-outxe2x80x9d vector, including identification of a complex mixture containing the clone of interest, long-range polymerase chain reaction (PCR) and ligation independent cloning. The present inventors are the first to generate a construct without isolating an individual genomic clone or mapping the restriction sites within the clone. Furthermore, the inventors are also the first to generate knock-out constructs using ligation independent cloning, including four-way annealing of nucleotide fragments. The subject invention provides novel constructs and efficient methods of making constructs which, when introduced into embryonic stem cells, deletes or mutates a specific gene in the target animal.
In one aspect, the invention includes a nucleotide construct comprising a sequence encoding a positive selection marker flanked by restriction enzyme sites. The restriction enzyme sites are flanked, on the side opposite the positive selection marker, by sequences which are not complementary to each other and which do not include one of the four types of base pairs at any position. The vector construct can be treated so that single-stranded regions are created at each sequence flanking one side of the restriction enzyme sites. More specifically, the nucleotide construct comprises a sequence encoding a positive selection marker flanked on each side by at least one restriction enzyme site. Preferably, the restriction enzyme site on each side of the positive selection marker is a unique site. Each of the aforementioned restriction enzyme site is flanked by a pair of annealing sites which do not contain at least one type of base at any position. The construct can be treated to create single-stranded regions and this creates the pair of annealing sites. None of the four annealing sites are complementary to each other so that when single-stranded regions are created, they cannot anneal to each other to reseal the vector, i.e., the single stranded regions are incompatible overhangs. However, the single stranded overhangs are compatible with, and can anneal to, the single stranded ends of insert fragments containing sequences homologous to the target gene or a target sequence. The restriction enzyme sites and annealing sites are designed for directional cloning.
Such a construct is illustrated, for example, in FIG. 2A which shows the plasmid pDG2. Plasmid pDG2 contains a unique restriction site, Sac II, between annealing sites 1 and 2 flanking one side of the positive selection marker (Neor in this case), and another unique restriction site, Sac I lying between annealing site 3 and site 4 flanking the other side of the positive selection marker.
In one embodiment, single-stranded regions are created by treating the vector with the appropriate restriction enzymes and with a DNA polymerase, for instance, T4 DNA polymerase. This procedure is described in detail in Example 1 below. In one embodiment, the construct comprises a plasmid vector and the positive selection marker is a neomycin resistance gene (Neor). Preferably, the screening marker on the side of the restriction enzyme sites outside the regions of the construct which are homologous to the target sequence, shown for example in FIG. 7, as opposite the positive selection marker. The screening marker can be green fluorescent protein (GFP) or a modified fluorescent protein.
In another embodiment, the construct of the present invention also includes a negative selection marker on the side of the restriction sites opposite the positive selection marker (e.g., next to the plasmid backbone sequences). The negative selection marker can be thymidine kinase (tk). However, unlike conventional targeted DNA constructs, the constructs described herein do not require, and are preferably made without, a negative selection marker.
In yet another preferred embodiment, the construct is the plasmid vector xe2x80x9cpDG2xe2x80x9d and has the sequence shown in SEQ ID NO:1. The construct can also be the plasmid vector xe2x80x9cpDG4, xe2x80x9d as shown in SEQ ID NO:2.
In another aspect, the invention provides a method of making a DNA construct useful in introducing a nucleotide sequence into a target DNA, comprising (a) amplifying a polynucleotide comprising two different nucleotide sequences substantially homologous to the target DNA; and (b) inserting a gene encoding for a positive selection marker between the two different nucleotide sequences substantially homologous to the target DNA. The positive selection marker may be, for example, a neomycin resistance gene (Neor). Preferably, the amplification step is performed in one-step from a genomic DNA library using, for example, oligonucleotide primers in a PCR reaction. In a preferred embodiment, the library is a plasmid library. In another embodiment, the amplified polynucleotide further comprises a gene encoding a selectable marker, for example, a gene encoding for ampicillin resistance. The vector can also include a second sequence coding for a screening marker, for example, green fluorescent protein (GFP), or another modified fluorescent protein.
In another aspect, the present invention also includes a method of making a DNA construct useful in introducing a nucleotide sequence into a target DNA, comprising: (a) providing a polynucleotide(s) substantially homologous to the target DNA; (b) generating two different fragments of the polynucleotide(s); (c) providing a vector having a gene encoding for a positive selection marker; and (d) using ligation independent cloning to insert the two different fragments into the vector to form the construct, wherein the positive selection marker is between the two different sequence fragments in the construct. The positive selection marker can be a neomycin resistance gene (Neor) and the vector may be pDG2 (SEQ ID NO:1) or pDG4 (SEQ ID NO:2). The vector can also include a second sequence coding for a screening marker, for example, green fluorescent protein (GFP) or another modified fluorescent protein. The vector can also include a second sequence coding for a negative selection marker.
In another embodiment, the method includes PCR amplifying the fragments with oligonucleotide primers having 5xe2x80x2 sequences which do not have one of the four base pairs at any position (also referred to herein as lacking one nucleotide). The 5xe2x80x2 sequences lacking one type of base are at least 5, preferably 12, even more preferably at least 20 to 25 nucleotides in length. In one embodiment, the oligonucleotide sequences are shown in SEQ ID NOs 3 to 10. In another embodiment, the oligonucleotide sequences are shown in SEQ ID NOs 3 to 44. The present invention also includes a method of making a DNA construct wherein the ligation independent cloning is performed in one step or in two steps.
The invention also provides a method of disrupting the function of a target sequence or gene in a cell by (a) inserting sequences homologous to the target gene into a construct of the invention as described above, such that the sequences homologous to the target gene flank the positive selection marker, to produce a targeting construct; and (b) introducing the targeting construct into the cell to produce a homologous recombinant wherein the function of the target gene or sequence is disrupted. In a preferred embodiment, the cell is an ES cell. A targeting construct produced by this method is also provided.
Another aspect of the invention is a method of enriching for the desired non-random integrant of the targeting vector wherein homologous recombination between the targeting vector and the target sequence or gene has mutated or disrupted the target gene. The enrichment step involves screening cells that have taken up the targeting construct, with ultraviolet light and identifying cells that do not fluoresce, for further testing by PCR or other methods to confirm the targeted mutation.
In yet another aspect, the invention includes a host cell or an animal containing a construct described herein. Where the construct is a targeting construct, preferably, the targeting construct disrupts the function of the target gene within the host cell or animal.
As will become apparent, preferred features and characteristics of one aspect of the invention are applicable to any other aspect of the invention.